Characterization and baculovirus-directed expression of a myosuppressin encoding cDNA from the true armyworm, Pseudaletia unipuncta.

Insect myosuppressins are a highly conserved sub-family of peptides which are primarily characterized by the ability to suppress contraction of visceral muscles in a variety of insect species. We have isolated a cDNA from the true armyworm, Pseudaletia unipuncta, that encodes a prohormone containing a peptide identical to ManducaFLRFamide. We have shown that this myosuppressin gene appears to be expressed in late larval and adult insects. In Manduca sexta, a number of extended-FLRFamide peptides have previously been purified including ManducaFLRFamide, F7D (DPSFLRFamide), F7G (GNSFLRFamide) and two larger peptides F24 and F39 that contain the shorter ManducaFLRFamide sequence at their C-terminus. Comparison with the true armyworm prepropeptide characterized here identifies F24 and F39 as partially processed products from the same precursor. Expression in the true armyworm was shown by in situ hybridization to occur in over 150 cells throughout the adult brain and nerve cord, and also to occur in both open and closed endocrine type cells of the gut. Overexpression of the P. unipuncta FLRFamide cDNA from a baculovirus vector in cabbage looper caterpillars was used to assess the potential for myosuppressin expression as a means of enhancing virus efficacy. Viral expression of the armyworm prohormone cDNA resulted in raised levels of RFamide-like products in the hemolymph of infected insects, but the products were found to be chemically distinguishable from authentic mature peptide and probably represent partially processed hormone.

Biological activities of the allatostatin family of peptides in the cockroach, Diploptera punctata, and potential interactions with receptors.

Allatostatins are a family of peptides that inhibit the production of juvenile hormone in the cockroach, Diploptera punctata. It is likely that the allatostatin prohormone precursor is processed to give rise to all 13 members of the family simultaneously. All members of the family show potency and efficacy, in terms of their ability to inhibit juvenile hormone production, albeit with dramatically different IC(50) and ED(50) values, ranging from a maximum of 0.014 nM for Dippu-AST 2 to 107 nM for Dippu-AST 1 (ED(50)). The likely occurrence of all 13 peptides in tissues and in haemolymph suggests that they may act in concert to produce physiological effects. We have employed combinations of the allatostatins, including a cocktail of all 13, 12 (minus Dippu-AST 2) and 11 (minus Dippu-AST 2 and 5) as well as mixtures of high and low activity allatostatins (Dippu-AST 5 plus either Dippu-AST 1 or 13) in dose-response studies to examine the possibility of synergistic or additive effects of the peptides on biological activity. None of the peptide combinations yielded evidence of synergistic interactions between allatostatins. However, the data do provide insight into receptor-ligand interactions in cockroaches and suggest the allatostatins regulate JH biosynthesis through a complex mix of differing affinity interactions with receptors in the corpora allata.

Allatostatins: a growing family of neuropeptides with structural and functional diversity.

The high degree of conservation of the core sequence of the "cockroach-types" of AST and their widespread distribution suggest that they should be considered a ubiquitous family of peptides within the invertebrates, regulating a range of important physiological processes. These functional processes, by either neural or humoral routes of action, include the inhibition of endocrine function, interneuronal functions, neuromodulatory roles, myotropic and myoendocrine roles, and direct action on biosynthetic pathways. The myomodulatory function appears to be conserved through evolutionary time, whereas the JH inhibitory activity appears to be confined to specific orders. This suggests that the myomodulatory role was the more ancestral of these two particular functions. Certainly, further purification and gene cloning as a means to precursor identification and functional analysis will be a prerequisite to understanding the diverse functions of this peptide family.

In situ hybridization analysis of leucomyosuppressin mRNA expression in the cockroach, Diploptera punctata.

In the cockroach Diploptera punctata, sequencing of the cDNA for the insect myoinhibitory neuropeptide, leucomyosuppressin (LMS), has demonstrated that LMS is the only Phe-Met-Arg-Phe-amide (NH2) (FMRFamide)-related peptide to be encoded by this gene (Donly et al. [1996] Insect Biochem. Mol. Biol. 26:627-637). However, in the present study, high performance liquid chromatography analysis of brain extracts showed six discrete FMRFamide-like immunoreactive fractions, one of which co-eluted with LMS. This study compared the distribution of FMRFamide-related peptides visualized by immunohistochemistry with LMS mRNA expression demonstrated by in situ hybridization in D. punctata. Immunohistochemistry with a polyclonal antiserum generated against FMRFamide, but which recognizes extended RFamide peptides, demonstrated numerous RFamide-like immunoreactive cells and processes in both nervous and nonnervous tissues. RFamide-like immunoreactivity was found in cells and processes of the brain and optic lobes, the stomatogastric nervous system, including the frontal and ingluvial ganglia, and the suboesophageal ganglion. Immunoreactivity was also present in all ganglia of the ventral nerve cord and in the alimentary canal. Within the alimentary canal, positively stained processes were found in the crop, midgut, and hindgut, and immunoreactive endocrinelike cells were located in the midgut. In situ hybridization with a digoxigenin-labeled RNA probe spanning the entire LMS coding region showed cell bodies containing LMS mRNA in all ganglia studied, other than the ingluvial ganglion. Expression was most abundant in the brain and optic lobes and in the frontal and suboesophageal ganglia. LMS mRNA was also apparent, although less intensely, in all other ganglia of the ventral nerve cord. Within the alimentary canal, LMS mRNA-positive cells were only visible in the anterior portion of the midgut, in the endocrinelike cells. The appearance of LMS mRNA in the central nervous system, stomatogastric nervous system, and midgut suggests that LMS may play a central role in Diploptera and may be associated with feeding and digestion.

Molecular cloning and functional expression of an insect high-affinity Na+-dependent glutamate transporter.

Excitatory amino acid transporters in the central and peripheral nervous systems of insects are thought to assist in maintaining glutamate concentrations in the resting synapse below the activation threshold of glutamate receptors. We have isolated a cDNA from the caterpillar Trichoplusia ni which encodes a high-affinity Na+-dependent glutamate transporter, designated TrnEAAT1. The deduced amino acid sequence shows strong identity with known members of the vertebrate Na+- and K+-dependent amino acid transporter family. Expression of the insect transporter mRNA was predominantly localized in the caterpillar brain. The function of the TrnEAAT1 protein was analyzed in cultured insect cells using a baculovirus expression system. Cells infected with the recombinant virus were found to exhibit a 50-fold increase in ability to accumulate labeled L-glutamate compared to mock-infected cultures, and this activity was shown to be Na+-dependent. Transport activity was further demonstrated by chromatographic identification of various glutamate analogues accumulated by infected cells. Various glutamate uptake inhibitors were used to outline the pharmacological properties of the cloned transporter and to compare it with known mammalian transporters. Despite the significant differences between insect and vertebrate physiology, the characteristics of the respective transporters were found to be remarkably similar.

Molecular characterization of the inhibitory myotropic peptide leucomyosuppressin.

The myoinhibitory peptide leucomyosuppressin (LMS) (pQDVDHVFLRFamide) has been identified and characterized at the molecular level in the cockroach Diploptera punctata through analysis of the organization of both brain cDNA and genomic DNA. Processing of the precursor predicted from DNA sequence would release a single LMS peptide. The organization of the precursor appears to be conserved in other insects and may reflect a functional organization for this subfamily of extended FLRFamides. The expression of the LMS gene appears in numerous cells of the pars-intercerebralis of the cockroach protocerebellum as well as in numerous endocrine cells of the midgut.

Characterization of the gene for leucomyosuppressin and its expression in the brain of the cockroach Diploptera punctata.

Using HPLC separation, radioimmunoassay, and subsequent bioassay, we have detected the presence of an active peptide, which co-elutes with the insect myoinhibitory peptide leuco-myosuppressin, in the brain of the cockroach Diploptera punctata. We have isolated a cDNA encoding the precursor for this peptide from cDNA libraries representing D. punctata brain RNA. The cDNA sequence contains an open reading frame that upon translation would result in a prepropolypeptide of 96 amino acids. Proteolytic cleavage of the predicted precursor could result in several peptides, including a 10 amino acid C-terminal peptide that would, upon modification of the NH2 and COOH-terminal amino acids, be identical to the insect FLRFamide, leucomyosuppressin. No other RFamide products are predicted to be processed from the precursor. Southern blot analysis indicates that the gene is present in the D. punctata genome in a single copy. Northern blot analysis shows that the gene is predominantly expressed as a 3.8 kb mRNA in cockroach brain. Study of the expression of the leucomyosuppressin gene in D. punctata brain, using in situ hybridization, indicates that expression occurs primarily in the pars intercerebralis of the protocerebrum, a region showing abundant FMRFamide-like immunoreactive neurosecretory cells. Immunohistochemistry and HPLC coupled to radioimmunoassay indicates that leucomyosuppressin represents a significant proportion of FMRFamide-related peptide production in the brain. However, HPLC analysis also indicates the presence of significant levels of other related peptides, demonstrating the presence of more than one FMRFamide-related gene in this insect.

Comparison of the allatostatin neuropeptide precursors in the distantly related cockroaches Periplaneta americana and Diploptera punctata.

Allatostatins are a family of insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have characterized cDNA and genomic DNA sequences that specify a preproallatostatin precursor in the oviparous cockroach Periplaneta americana. Comparison of this precursor with that previously described [Donly, B. C., Ding, Q., Tobe, S. S. & Bendena, W. G. (1993) Proc. Natl Acad. Sci. USA 90, 8807-8811] for the viviparous cockroach Diploptera punctata revealed several common features. First, the precursors are remarkably similar in size and the organization of the peptides within the precursor is conserved. The separation of the peptides into groups by acidic domains within the precursor has been maintained. The P. americana precursor contains 14 allatostatin-like peptides that contain the core C-terminal sequence (Tyr/Phe)-Xaa-Phe-Gly-(Leu/Ile)-NH2, as compared to the D. punctata precursor, which contains 13. Five of the peptides are perfectly conserved between the two species. The remainder, with one exception, contain amino acid substitutions in the N-terminal address portion of the peptide. Several features of expression are also similar between these two species. In both, a single copy gene specifies a large allatostatin transcript of 5.0 kb in P. americana and 9.2 kb in D. punctata. In P. americana, allatostatin transcripts appear to be produced by numerous cells in different regions of the brain.

Competition between frameshifting, termination and suppression at the frameshift site in the Escherichia coli release factor-2 mRNA.

Competition between frameshifting, termination, and suppression at the frameshifting site in the release factor-2 (RF-2) mRNA was determined in vitro using a coupled transcription-translation system by adding a UGA suppressor tRNA. The expression system was programmed with a plasmid containing a trpE-prfB fusion gene so that each of the products of the competing events could be measured. With increasing concentrations of suppressor tRNA the readthrough product increased at the expense of both the termination and the frameshifting product indicating all three processes are in direct competition. The readthrough at the internal UGA termination codon was greater than that at the natural UGA termination codon at the end of the coding sequence. The results suggest that this enhanced suppression may reflect slower decoding of the internal stop codon by the release factor giving suppression a competitive advantage. The internal UGAC stop signal at the frameshift site has been proposed to be a relatively poor signal, but in addition the release factor may be less able to recognise the signal with the mRNA in such a constrained state. Consequently, the frameshifting event itself will be more competitive with termination in vivo because of this longer pause as the release factor is decoding the stop signal.

Molecular cloning of the gene for the allatostatin family of neuropeptides from the cockroach Diploptera punctata.

Allatostatins (ASTs) are insect neuropeptides that inhibit juvenile hormone biosynthesis by the corpora allata. We have isolated a cDNA from the cockroach Diploptera punctata that encodes a 41.5-kDa precursor polypeptide containing the AST family of peptides. Translation of the cDNA revealed a 370-amino acid pre-pro-peptide consisting of 13 AST-type peptides and appropriate processing sites for endoproteolytic cleavage and amidation. The 13 potential AST sequences are characterized by the C-terminal AST corestructure Phe-Gly-Leu-NH2, with only one exception. Separating the clustered ASTs in the precursor, three acidic spacer regions are found. Contained within the largest of these are two potentially related peptides that may also be processed. Southern blot analysis revealed the presence of a single copy of the AST gene per haploid genome, as well as the probability that the gene may be present in at least two allelic forms. In situ hybridization indicated the AST-encoding gene is expressed in neurosecretory cells of D. punctata brain.

Functional domains in the Escherichia coli release factors. Activities of hybrids between RF-1 and RF-2.

Chimeras between Escherichia coli release factors RF-1 and RF-2 have been constructed to study the role of the release factors in termination, in particular whether each possesses specific domains for recognition of the stop codon, and for facilitating peptidyl-tRNA hydrolysis. One hybrid factor showed normal codon-recognition activity but was defective in its ability to facilitate hydrolysis. Overexpression of this protein was toxic to the cell. Conversely, another hybrid factor showed complete loss of codon recognition but retained some hydrolysis activity. These two functional activities of the release factors were not localised in domains within either the amino-terminal or carboxy-terminal halves of the primary sequence as previously predicted. Evidence from the activities of the hybrid proteins and from earlier studies suggests that a combination of residues from the beginning and middle of the sequence, including a region of very high sequence conservation, contribute to the hydrolysis domain, whereas residues from both the amino-terminal and carboxy-terminal halves of the molecule are important for the codon recognition domain.

Frameshifting by eukaryotic ribosomes during expression of Escherichia coli release factor 2.

Normal translation of the gene for E. coli release factor 2 (RF-2) is characterized by a +1 frameshift event that occurs with 30-50% efficiency. Frameshifting on synthetic RF-2 mRNA by eukaryotic ribosomes has also been observed, even though they lack the capability to interact with the frameshift signal in the same manner as prokaryotic ribosomes. We have mutagenized the sequence of the RF-2 gene to eliminate the need for a frameshift, thereby allowing frameshifting efficiency to be measured by direct comparison of RF-2 production from the mutant with production from the wild-type. Measurements using this approach confirm that frameshifting by rabbit reticulocyte lysate ribosomes occurs at the frameshift region, but with a limited efficiency of approximately 0.4%.

Frameshift autoregulation in the gene for Escherichia coli release factor 2: partly functional mutants result in frameshift enhancement.

The regulation of release factor 2 (RF-2) synthesis in Escherichia coli occurs, at least in part, through autoregulatory feedback exerted at a unique frameshifting step required during RF-2 translation. We have constructed fusions between the genes for RF-2 and E. coli trpE which make direct measurement of frameshifting efficiency possible since both products of regulation, the termination product and the frameshift product, are stable. The addition of purified RF-2 to in vitro expressions of these fusion genes was found to result in decreased frameshifting and increased termination at the regulation site. The frame-shifted trpE-RF-2 products synthesized from these fusions are unique with respect to their functional release factor activities; when tested in assays of two intermediate steps of translational termination, they were found to be partially active for the function of ribosome binding, but inactive for peptidyl-tRNA hydrolysis (release). These are the first examples of release factor mutants selectively active for only one of these function. In vivo these chimeric proteins promote large increases in frameshifting at the RF-2 frameshift region, thereby reversing normal negative autoregulatory feedback and instead supporting fully efficient frameshifting in their own synthesis. This activity provides new evidence for the importance of ribosomal pausing in directing efficient frameshifting at the RF-2 frameshift region.

Tightly controlled expression systems for the production and purification of Escherichia coli release factor 1.

The genes for the protein release factors in Escherichia coli have traditionally proven difficult to maintain on high copy plasmids. We have established here systems which provide for both stable maintenance of the release factor 1 gene on such plasmids, as well as high level overproduction of the release factor 1 protein. The gene is maintained under the control of the inducible trc or tac promoters in the presence of very high levels of lac repressor. A simple and rapid scheme for the purification of RF1 from extracts of cultures carrying these plasmids is also described.

Frameshifting in the synthesis of Escherichia coli polypeptide chain release factor two on eukaryotic ribosomes.

A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. The nucleotide sequence preceding the in-phase stop codon within RF-2 mRNA is complementary to the 3' anti-(Shine-Dalgarno sequence) region found in prokaryotic 16S rRNA and Weiss et al. (1988) have concluded that this pairing triggers the frameshift event. In vitro production of RNA coding for RF-2, suitable for translation on eukaryotic ribosomes, has enabled testing of whether eukaryotic ribosomes can frameshift at this sequence. The 18S rRNA of eukaryotic ribosomes does not contain the 3' anti-(Shine-Dalgarno sequence) region. The prokaryotic RF-2 gene and the gene for the other release factor, RF-1, which does not contain an in-frame stop codon, were subcloned into transcription vectors such that the RNA transcripts produced in vitro would resemble a typical eukaryotic mRNA. These RF-1 and RF-2 RNAs both synthesized a major product of Mr approximately 45,000 when translated in vitro within reticulocyte lysate; the size expected for full length RF-1 and RF-2 molecules. The RF-2 product was immunoprecipitated by RF-2-specific antibodies, including those to regions of the protein encoded in the mRNA downstream from the frameshift site. The putative premature termination product, an oligopeptide of 25 amino acids, was not detected, but a chemically synthesized derivative was shown to be very unstable within the translation system. Although it was not possible therefore to calculate an absolute efficiency of frameshifting, the relative efficiency of the translation of RF-2 RNA was estimated to be 10-20% of that of RF-1 RNA in the reticulocyte system. This was similar to the relative synthesis of the two proteins in a plasmid-DNA-directed prokaryotic transcription/translation system. These results show that in vitro on eukaryotic ribosomes where the Shine-Dalgarno-type interaction is not possible, high efficiency frameshifting around the in-phase stop codon in the RF-2 mRNA can still occur.

Mutations in the leader sequence and initiation codon of the gene for ribosomal protein S20 (rpsT) affect both translational efficiency and autoregulation.

We have transferred the complete structural gene and part of the leader for ribosomal protein S20 of Escherichia coli to a controllable expression vector and have used oligonucleotide-directed mutagenesis to create mutations in the untranslated leader of the plasmid-borne gene. We have assayed for posttranscriptional regulation of the synthesis of S20 after inducing transcription of the mutant S20 mRNA from the expression vector. We found that two mutations lead to loss of feedback control of S20 synthesis: (i) a change of the initiation codon from UUG to AUG and (ii) a replacement of part of the S20 leader with a nonhomologous sequence including an AUG initiation codon. These mutations also lead to increases in both the intrinsic translational efficiency of the plasmid-encoded S20 mRNA in vitro and its half-life in vivo. A double mutation (GA to CT) at residues -3 and -4 relative to the initiation codon does not result in overproduction of S20. Rather, it reduces translational efficiency in vitro and mRNA stability in vivo. Our results demonstrate the fundamental importance of the UUG initiation codon in mediating autogenous repression of S20 synthesis.

Affinities of ribosomal protein S20 and C-terminal deletion mutants for 16S rRNA and S20 mRNA.

We have measured the binding of E. coli ribosomal protein S20 and a number of C-terminal deletion mutants to 16S rRNA and in vitro transcribed S20 mRNA. Mutant S20s of interest were synthesized in vitro from the appropriate plasmid templates by coupled transcription and translation. The affinity of S20 produced in vitro for 16S rRNA is 1.2 x 10(7) (M-1) in a gel filtration assay. Removal of as few as 6 residues from the C terminus of S20 results in a sharp loss of binding activity, suggesting the presence of critical residues in this region. Analysis of the amino acid sequence of S20 indicates that these residues may constitute part of a segment of alpha helix. Although S20 is known to autoregulate its own synthesis, we were unable to demonstrate any measurable affinity of S20 for its own mRNA.